Precisely regulated expression of photoreceptor-specific genes is essential for the development and survival of photoreceptors. The ultimate goal of our research is to identify the molecular mechanisms regulating the expression of these genes in the mammalian retina and to determine how disruption of these mechanisms causes photoreceptor disease. A network of photoreceptor transcription factors is responsible for this regulation, and a key member of this network is the cone-rod homeobox protein (Crx). CRX mutations cause photoreceptor degeneration in humans. To understand the mechanism of action of Crx, several Crx interacting proteins have been identified and their functions are being investigated. These include three ubiquitously expressed co-activators, Gcn5, Cbp and p300, which modulate chromatin conformation via their histone acetyltransferase (HAT) activity. Our preliminary data suggest that these co-activators are important components of the Crx regulatory pathway. We hypothesize that the concerted actions of Crx and its co-activators modulate chromatin conformation to regulate the expression of photoreceptor genes during retinal cell development and survival. This hypothesis will be tested in two specific aims. In Aim 1 we will determine the role of the three HATs in photoreceptor development and survival using conditional knockout mouse models. Mice carrying floxed alleles of each co-activator are being crossed with transgenic mice expressing Cre in developing (Crx-Cre) or mature (Rho-iCre) photoreceptors. In cells that express Cre, the floxed genes will be knocked out. Phenotypes of the resulting mutant mice are analyzed by morphological, biochemical and functional assays. As a complementary approach, mutant co-activators will be ectopically expressed in Y79 retinoblastoma cells and neonatal mouse retinas to determine whether the function of the co-activators depends on their HAT catalytic activity or their interaction with Crx. Together, these molecular genetic approaches will reveal the role and mechanism of action of each co-activator in the Crx regulatory pathway. In addition to histone acetylation, several other forms of epigenetic modulation, including histone methylation and intrachromosomal interactions, also regulate transcription. Aim 2 is designed to test the hypothesis that differential epigenetic modulation regulates the transcription of rod and cone genes in individual cells, thereby determining photoreceptor subtypes. Chromatin immunoprecipitation (ChIP) assays on isolated, sorted rods or cones from mouse retinae will be used to correlate the histone modifications on rod and cone opsin genes with transcriptional activation or repression in each photoreceptor subtype. Chromosome conformation capture (3C) assays will reveal intrachromosomal interactions between different regions of the opsin genes and their importance in transcription. This study represents the first comprehensive epigenetic investigation in the retina. The findings will significantly advance our understanding of photoreceptor gene expression in retina development, maintenance and disease. PUBLIC HEALTH RELEVANCE: Crx is a protein that regulates the expression of many genes important for photoreceptor function. Crx mutations cause photoreceptor degeneration diseases that lead to incurable blindness. This research investigates how Crx interacts with other proteins to keep photoreceptors healthy, and why mutations make photoreceptors sick, providing new insight into therapy development.